1. Hepatic rRNA Transcription Regulates High-Fat-Diet-Induced Obesity
Shohei Oie, Kazuya Matsuzaki, Wataru Yokoyama, Shinji Tokunaga, Tsuyoshi Waku, Song-Iee Han, Naoya Iwasaki, Aya Mikogai, Kayoko Yasuzawa-Tanaka, Hiroyuki Kishimoto, Hiromi Hiyoshi, Yuka Nakajima, Toshiyuki Araki, Keiji Kimura, Junn Yanagisawa, Akiko Murayama 
Cell Reports 
Volume 7, Issue 3, Pages (May 2014)
DOI: /j.celrep
Copyright © 2014 The Authors Terms and Conditions

2. Cell Reports 2014 7, 807-820DOI: (10.1016/j.celrep.2014.03.038)
Copyright © 2014 The Authors Terms and Conditions

3. Figure 1
Hepatic Pre-rRNA Transcription Is Repressed in Obese Mice Fed on High-Fat Diet
(A) Body weight of WT (C57BL6) fed on normal chow diet (NC), high-fat diet 1 (HFD1) for 8 weeks, and HFD2 for 24 weeks.
(B) Pre-rRNA levels in livers of WT mice fed on NC, HFD1 for 8 weeks, and HFD2 for 24 weeks by quantitative RT-PCR (qRT-PCR). Pre-rRNA levels were normalized to cyclophilin.
(C) Body weight of WT and obesity model mice (ob/ob mice) fed on NC at the age of 24 weeks.
(D) Pre-rRNA levels in livers of WT and ob/ob mice fed on NC at the age of 24 weeks by qRT-PCR. Pre-rRNA levels were normalized to cyclophilin.
(E) The structure of the mouse rRNA gene. Chromatin immunoprecipitation (ChIP) assay was performed with DNA extracted from immunoprecipitates with primer pairs that recognize sequences within the over the promoter regions (M0) and the downstream of the transcribed region (M9).
(F–I) ChIP assay in WT livers fed on either NC or HFD1 for 8 weeks using normal immunoglobulin G (IgG), anti-H3 antibody, anti-H3K9me2 antibody (F), anti-H3Ace antibody (G), anti-NML antibody (H), and anti-SIRT1 antibody (I). Amounts of H3K9me2 and H3Ace are expressed relatively to the signal obtained for ChIP using H3, and amounts of NML and SIRT1 are expressed relatively to the signal obtained for ChIP using IgG.
(J) Expression levels of NML and SIRT1 in WT livers fed on either NC or HFD1 for 8 weeks by immunoblotting.
All values are presented as mean ± SEM. n = 3–8; ∗p < See also Figure S1.
Cell Reports 2014 7, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

4. Figure 2 NML Regulates Pre-rRNA Transcription in the Liver
(A) Pre-rRNA levels in liver, brown adipose tissue (BAT), white adipose tissue (WAT), and skeletal muscle (SM) of WT and NML-KO mice by qRT-PCR. Pre-rRNA levels were normalized to cyclophilin. n.s., not significant.
(B) Expression levels of NML, SIRT1, and Suv39h1 in liver, BAT, WAT, and SM of WT and NML-KO mice by immunoblotting.
(C–F) ChIP assay in WT and NML-KO livers using normal IgG, anti-H3 antibody, anti-H3K9me2 antibody (C), anti-H3Ace antibody (D), anti-SIRT1 antibody (E), and anti-Pol I antibody (F). Amounts of H3K9me2 and H3Ace are expressed relatively to the signal obtained for ChIP using H3, and amounts of SIRT1 and Pol I are expressed relatively to the signal obtained for ChIP using IgG.
(G) The AMP and ATP contents in WT and NML-KO livers by high-performance liquid chromatography (HPLC).
(H) Protein levels of phosphorylated AMPK and total AMPK in WT and NML-KO livers by immunoblotting.
All values are presented as mean ± SEM. n = 3–22; ∗p < 0.05 and ∗∗p < n.s., not significant. See also Figure S2.
Cell Reports 2014 7, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

5. Figure 3
NML Deficiency in the Liver Affects Lipid-Metabolism-Related Gene Expression
(A) Ingenuity pathway analysis predicts nine different pathways from the analysis of gene-expression profiles of WT and NML-KO livers using Affymetrix microarray technology.
(B) The expression levels of fatty acid oxidation (FAO)- and mitochondrial-activity-related genes in WT and NML-KO livers by qRT-PCR. Each mRNA level was normalized to cyclophilin.
(C) The expression of de novo lipid synthesis of fatty-acids-related genes in WT and NML-KO livers by qRT-PCR. Each mRNA level was normalized to cyclophilin.
(D) The expression levels of glycolysis- and gluconeogenesis-related genes in WT and NML-KO livers by qRT-PCR. Each mRNA level was normalized to cyclophilin.
All values are presented as mean ± SEM. n = 3–9; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < See also Figure S3.
Cell Reports 2014 7, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

6. Figure 4
NML Deficiency in Hepatocytes Regulates Hepatic Lipid Metabolism
(A) Pre-rRNA levels in primary hepatocytes isolated from the WT and NML-KO mice by qRT-PCR. Pre-rRNA levels were normalized to cyclophilin.
(B) The rate of protein synthesis in the primary hepatocytes isolated from the WT and NML-KO mice was measured by the amount of [35S]-methionine incorporated into protein.
(C) The enzymatic activity of AMPK in the primary hepatocytes isolated from the WT and NML-KO mice using the SAMS peptide assay.
(D) FAO rate in the primary hepatocytes isolated from the WT and NML-KO mice was measured by oxidation [14C]-oleate into [14CO2].
(E) The expression levels of FAO- and mitochondrial-activity-related genes in the primary hepatocytes isolated from the WT and NML-KO mice by qRT-PCR. Each mRNA level was normalized to cyclophilin.
(F) De novo fatty acid synthesis rate in the primary hepatocytes isolated from the WT and NML-KO mice was measured by the amount of [14C]-acetate incorporated into fatty acids.
(G) The expression of de novo lipid synthesis of fatty-acids-related genes in the primary hepatocytes isolated from the WT and NML-KO mice by qRT-PCR. Each mRNA level was normalized to cyclophilin.
(H) Mitochondrial oxygen consumption in the primary hepatocytes isolated from the WT and NML-KO mice using the XF24 analyzer. During the real-time measurement, respiratory chain inhibitors (oligomycin, 2,4-DNP, and rotenone) were added to the culture at the indicated time points. The maximal respiratory capacity of the mitochondria indicates the difference in OCR between 2,4-DNP- and rotenone-added states.
(I) Mitochondrial DNA contents in the primary hepatocytes isolated from the WT and NML-KO mice by quantitative PCR.
All values are presented as mean ± SEM. n = 3–9; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < See also Figure S4.
Cell Reports 2014 7, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

7. Figure 5
NML Deficiency Prevents High-Fat-Diet-Induced Hepatic Steatosis
(A) Pre-rRNA levels in the livers of WT and NML-KO mice fed on either NC or HFD1 for 8 weeks by qRT-PCR (n = 5–20). Pre-rRNA levels were normalized to cyclophilin.
(B) Morphology of liver sections of WT and NML-KO mice fed on either NC or HFD1 for 8 weeks.
(C) Representative of liver steatosis using oil red O staining of liver sections in WT and NML-KO mice fed on either NC or HFD1 for 8 weeks.
(D–F) Hepatic triglyceride (D), total cholesterol (E), and total free fatty acid (TFFA) (F) contents of WT and NML-KO mice fed on either NC or HFD1 for 8 weeks (n = 6–10).
(G) The expression levels of PGC1α and CPT1 genes in WT and NML-KO livers fed on HFD1 for 8 weeks by qRT-PCR (n = 6–12). Each mRNA level was normalized to cyclophilin.
(H) The expression levels of SREBP1c, SCD1, and FAS genes in WT and NML-KO livers fed on HFD1 for 8 weeks by qRT-PCR (n = 10). Each mRNA level was normalized to cyclophilin.
All values are presented as mean ± SEM. ∗p < 0.05 and ∗∗p < See also Figure S5.
Cell Reports 2014 7, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

8. Figure 6
NML Deficiency Increases Whole-Body Energy Expenditure and Prevents High-Fat-Diet-Induced Obesity
(A) Growth curve of the WT and NML-KO mice fed on either NC or HFD1 for 8 weeks (n = 11–15).
(B) Left: Computed tomography (CT) scanning of the WT and NML-KO mice fed on either NC or HFD1 for 8 weeks. Middle: Visceral fat areas of the WT and NML-KO mice fed on either NC or HFD1 for 8 weeks. Right: Subcutaneous fat areas of WT and NML-KO mice fed on either NC or HFD1 for 8 weeks (n = 9–14).
(C) The epididymal fat mass of the WT and NML-KO mice fed on either NC or HFD1 for 8 weeks (n = 8–11).
(D–G) Serum TG (D), total cholesterol (E), TFFA (F), and glucose (G) contents of the WT and NML-KO mice fed on either NC or HFD1 for 8 weeks (n = 3–15).
(H) Food intake in the WT and NML-KO mice fed on either NC or HFD1 for 8 weeks (n = 3–5).
(I) O2 consumption in the WT and NML-KO mice fed on either NC or HFD1 for 8 weeks (n = 4–5).
(J) Locomotor activity in the WT and NML-KO mice fed on either NC or HFD1 for 8 weeks (n = 4–5).
(K) Body temperature in the WT and NML-KO mice fed on either NC or HFD1 for 8 weeks (n = 3–6).
All values are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < See also Figure S5.
Cell Reports 2014 7, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

9. Figure 7
Liver-Specific NML Deficiency Prevents High-Fat-Diet-Induced Obesity
(A) Expression levels of NML in liver, BAT, WAT, and SM of the control and liver-NML-KO mice by immunoblotting.
(B) Pre-rRNA levels in the control and liver-NML-KO livers fed on NC by qRT-PCR (n = 7). Pre-rRNA levels were normalized to cyclophilin.
(C) The AMP/ATP ratio in the control and liver-NML-KO livers fed on NC by HPLC (n = 3–4).
(D) Protein levels of phosphorylated AMPK and total AMPK in the control and liver-NML-KO livers fed on NC by immunoblotting.
(E and F) The expression levels of FAO-, mitochondrial-activity-related (E), and de novo lipid synthesis of fatty-acids-related genes (F) in the control and liver-NML-KO livers by qRT-PCR (n = 3–14). Each mRNA level was normalized to cyclophilin.
(G) Growth curve of the control and liver-NML-KO mice fed on HFD1 for 8 weeks (n = 8–10).
(H) Left: CT scanning of the control and liver-NML-KO mice fed on HFD1 for 8 weeks. Right: Visceral fat areas of the control and liver-NML-KO mice fed on HFD1 for 8 weeks (n = 3–4).
(I) The epididymal fat mass of the control and liver-NML-KO mice fed on HFD1 for 8 weeks (n = 6–7).
(J) O2 consumption in the control and liver-NML-KO mice fed on either NC or HFD1 for 8 weeks (n = 4–5).
(K and L) Hepatic TG (K) and TFFA (L) contents of the control and liver-NML-KO mice fed on HFD1 for 8 weeks (n = 6–8).
All values are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < See also Figure S6.
Cell Reports 2014 7, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions